Fig. 2

Silencing miR-21 reduces ROS and ameliorates mitochondrial membrane potential (ΔΨm) in oscillating (OG) and high stable glucose (HG) conditions. a Representative images of oxidized MitoSOX Red staining using fluorescence microscopy. Cells imaged by fluorescence microscope using a rhodamine filter (peak excitation 547 nm, emission filter 580 nm long pass). Arrows show the major fluorescence intensity peak for each image. b Quantification of MitoSOX images measured the % of MitoSOX fluorescence intensity area/count of nuclei (DAPI) per field (at least n = 6 per condition). The red intensity (arbitrary fluorescence units equivalent to mitochondrial superoxide production) was quantified, integrated, and normalised to integrated DAPI intensity (equivalent to cell number) using ImageJ. Scale bar, 20 μm. *p < 0.05 and **p < 0.01 versus NG, ^††††p < 0.0001 vs OG and ^§§§§p < 0.0001 vs HG. c Selective detection of mitochondrial superoxide anion using MitoSOX Red staining by FACS analysis (n = 3 set experiments) expressed as a % of the max fluorescence intensity. d Densitometry of FACS as a fold change of the fluorescence intensity. **p < 0.01 and ****p < 0.0001 vs NG, ^§p < 0.05 vs HG. e Representative images of fluorescence microscopy show mitochondrial membrane potential (ΔΨm) changes. Red staining indicates JC-1 aggregates in control cells (NG), revealing intact mitochondria, and the formation of monomers (green staining) shows dissipation of ΔΨm. A decrease in monomers and increase in aggregates (red staining) indicate the ability of anti-miR-21 to protect the mitochondria. f The graph under the images indicates the ratio of JC-1 aggregates to JC-1 monomers calculated by ImageJ software (http://www.imagej.nih.gov, ImageJ, NIH, Bethesda, MD, USA). The results are expressed as mean (± SEM) of the ratio of arbitrary unit intensity, n = 6 and ***p < 0.001 vs NG; ^†p < 0.05 vs OG, ^†††p < 0.001 vs OG; ^§§§p < 0.001 vs HG. Symbols over the bars refer to differences between the conditions shown under the bars